Method and means for producing high titer, safe, recombinant lentivirus vectors

Abstract

Lentiviral vectors modified at the 5' LTR or both the 5' and 3' LTR's are useful in the production of recombinant lentivirus vectors. Such vectors can be produced in the absence of a functional tat gene. Multiple transformation of the host cell with the vector carrying the transgene enhances virus production.

We claim:  
     
       1. A lentivirus packaging plasmid lacking sequences upstream from gag endogenous to said lentivirus and lacking sequences downstream from env endogenous to said lentivirus. 
     
     
       2. The packaging plasmid of  claim 1 , wherein said packaging plasmid comprises a gag, a pol or gag and pol genes. 
     
     
       3. The packaging plasmid of  claim 1 , wherein said packaging plasmid carries a non-functional tat gene. 
     
     
       4. The packaging plasmid of  claim 1 , wherein said lentivirus is human immunodeficiency virus (HIV). 
     
     
       5. The packaging plasmid of  claim 4 , wherein said HIV is HIV-1. 
     
     
       6. A method for producing a recombinant lentivirus vector comprising: 
       a) transforming a cell with:  
       i) at least one lentivirus packaging plasmid lacking sequences upstream from gag endogenous to said lentivirus and lacking sequences downstream from env endogenous to said lentivirus, and said at least one packaging plasmid comprises a gag, a pol or gag and pol genes; and  
       ii) an expression plasmid not endogenous to said lentivirus which carries an env gene not endogenous to said lentivirus; to yield a packaging cell;  
       b) multiply transforming said packaging cell with a lentivirus transfer vector which comprises a heterologous gene to yield a producer cell;  
       c) culturing said producer cell in a medium; and  
       d) separating said producer cell from said medium to recover said recombinant lentivirus vector from said medium.  
     
     
       7. The method of  claim 6 , wherein said packaging cell carries a non-functional tat gene. 
     
     
       8. The method of  claim 6 , wherein said lentivirus transfer vector comprises a 5′ LTR and a 3′ LTR, each of which contains a U3 region, wherein a part or all of a regulatory element of the U3 region of the 5′ LTR is replaced by another regulatory element, operable in a mammalian cell, which is not endogenous to said lentivirus. 
     
     
       9. The method of  claim 8 , wherein one or more nucleotide bases of the U3 region of the 3′ LTR are deleted.